The overall objective of this proposal is to develop and combine two entirely new experimental genomic technologies for identifying functional regulatory elements in non-coding DMA. The first is a genome-wide method for identifying protein-binding loci in any sequenced genome. It is based on high-throughput sequence-tag analysis of DMA that is recovered after Chromatin |mmunoPrecipitation (ChIP). We term this method STAGE, for Sequence Tag Analysis of Genomic Enrichment. STAGE can overcome many of the limitations of promoter/intergenic microarrays that are currently under development for the human and other large genomes. However, the utility of STAGE for this purpose is constrained by the ChIP technique, which must be performed one protein at a time and suffers from other limitations such as the quality and availability of antibodies to proteins of interest. Therefore, the second method we propose to develop is a procedure for biochemically purifying and identifying all potential regulatory elements from total chromatin without the requirement for antibodies or affinity tags (FAIRE, for Formaldehyde-Assisted isolation of Regulatory Elements). FAIRE makes high-throughput characterization of regulatory elements possible by taking advantage of a chromatin feature common to all active regulatory elements (namely, nucleosome clearance) instead of using specific proteins to isolate them. Thus FAIRE can overcome the limitations of chromatin immunoprecipitation. We will develop this method for use in complex genomes such as C. elegans and human. In order to demonstrate the utility of FAIRE beyond yeast, an unbiased detection method like STAGE is required. Conversely, FAIRE is ChlP-independent, and therefore can be used to cross-validate ChlP-based STAGE results. This natural synergy between our two independent novel techniques makes each one more powerful and has the potential to accelerate the identification of protein binding sites and regulatory elements in any sequenced genome.